1. Field of the Invention
The present invention relates to a method of staining, and detecting and counting bacteria in clinical samples, in particular, bacteria in urine samples, and a diluent for bacterial stain.
2. Related Art
The number of bacteria in urine is an important parameter in clinical diagnosis to judge the presence of infection. In general, the presence of bacteria of 105 or more/ml in urine is recognized as a criterion of positive urinary tract infection. If urine contains bacteria of 103 or more/ml, it is diagnosed as contaminated urine (normal bacteria flora), i.e., negative urinary tract infection. If bacteria of about 104/ml is observed, the diagnosis is reserved but the sample is often re-examined.
Conventionally, observation of bacteria in urine has been performed by microscopic examination of Gram stained bacteria, unstained bacteria without Gram staining treatment or fluorescence-stained bacteria.
Urine often contains contaminants such as mucus threads, crystals, amorphous salts and cell fragments that are clinically insignificant. These substances hinder the measurement of significant particles (in particular bacteria) so that it has been difficult to accurately count the number of bacteria. Actually, there has been no method of counting bacteria of about 104/ml, accurately.
In the case of Gram stain, bacteria and contaminants are stained simultaneously so that counting loss of bacteria of a small number occurs frequently in the microscopic examination. Further, Gram stain includes a number of staining steps and takes time (about 15 minutes) so that working efficiency is poor.
The microscopic examination of bacteria without staining treatment can be carried out quickly, but it cannot discriminate bacteria particularly when coccus contaminants are contained.
The microscopic examination of fluorescence-stained bacteria shows better detectability than the above-mentioned two methods. However, there has not been established how to eliminate other contaminants than bacteria and to stain the bacteria quickly.
Agar medium method, which is a standard method, requires 16 hours or more to determine the bacteria number, so that it cannot be regarded as a quick method.
U.S. Pat. No. 4,622,298, and Japanese Unexamined Patent Publication No. Hei 9 (1997)-119926 and No. Hei 9 (1997)-329596 each proposes a method of detecting bacteria in a fluorescence-stained urine sample with a flow cytometer.
A polymethine dye utilizes for fluorescence staining in the above references, but some bacteria are not sufficiently stained with the polymethine dye. For example, in the case of a sample in which nitrate-reducing bacteria proliferate and produce a large amount of nitrite, nitrite ions decompose the polymethine dye so that the dye does not effectively work on the bacteria staining.
Usually, bacteria are stained well at acidic pH. Further, a urine sample which contains mucus threads is effective in the bacteria staining. However, effect of the nitrite ions is promoted at acidic pH.